Two decades ago, we identified abnormalities of rab5-positive endosomes as a signature pathology of Alzheimer?s disease (AD), which is considered the earliest appearing neuronal pathology specific to AD yet known. Genetic data have independently converged on endocytosis as a prime early target in AD. We have shown that development of endosome anomalies requires pathological hyper-activation of rab5, the master regulatory GTPase on early endosomes, to trigger markedly upregulated endocytosis and aberrant endosomal fusion, trafficking, and cell signaling leading to cholinergic neurodegeneration. Rab5 over-activation is caused by elevated endosomal levels of APP-CTF (-cleaved C-terminal fragment of APP) in AD and in Down Syndrome (DS), a cause of AD due to an extra APP copy. APP-CTF directly binds and recruits APPL1, a signaling effector, which aberrantly stabilizes the activated (GTP-loaded) state of rab5, triggering endosomal dysfunction. The pathogenic importance of rab5 over-activation is underscored by the phenotype of our new transgenic mouse model of neuronal rab5 over-activation, which develops AD-related endosome dysfunction and AD-like deficits in retrograde transport, trophin signaling, synaptic plasticity, cognition, and neuron survival, all reflecting impairment of rab5?s many known roles in neurons. We propose that early endosome dysfunction is a key part of the antecedent pathobiology initiating -amyloidogenesis in late-onset AD and that its underlying basis, rab5 hyper-activation, further drives AD development through multiple pathways. We further propose that other genetic influences increasing AD risk, including ApoE and GWAS-identified genes with roles in endocytosis increase disease risk by dysregulating rab5 or exacerbating rab5?s impact on endosome dynamics and cell signaling. To validate these concepts, we will define the multiple pathways/factors that regulate rab5 activity in neurons (Aim1a) and that initiate AD-related endosome dysfunction via rab5 and additional factors controlling endosomal recycling and maturation in cell and mouse models of AD and DS (Aim 1b). We will define the consequences of selective rab5 over-activation on brain function in vivo and validate its predicted disease- accelerating effects in an hAPP(wt) mouse model of AD(Aim 2). Of exciting clinical relevance, we will validate in vivo the predicted actions on endocytosis of an AD therapeutic in current clinical trials, which we have shown to reverse rab5 activation and endosome dysfunction in DS patient cells at low nanomolar concentrations (Aim 3). The crucial roles of APPL1 and APPL2 in directly linking APP- CTF to rab5 hyper-activation will be explored and also validated in our mice that over-express APPL1 or lack APPL1 and/or APPL2 and also in these mice crossed to AD models (Aim 4). We will test the hypothesis that GWAS AD risk genes with suspected roles in endocytosis confer risk in part by exacerbating rab5-driven endosome dysfunction (Aim 5). These are all novel unexplored areas of AD pathobiology that are highly relevant to the molecular origin(s) of AD. Our ultimate objectives are identifying and validating innovative therapeutics that target this earliest AD pathobiology.